Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/808—Optical sensing apparatus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/805—Optical property
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/11—Automated chemical analysis
  • Y10T436/115831—Condition or time responsive
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

• HTS high throughput screening
• UHTS ultra-high throughput screening
• Denaturation of proteins is accompanied by the progressive loss of their tertiary/quaternary structure and ultimately biological activity.
• Denaturation can be accomplished by a number of physical and chemical methods that involve changes in temperature, pH, and/or, ionic strength, use of chaotropic agents, etc. It can be followed by methods sufficiently sensitive to monitor conformational changes in a protein. Because it is a simple and widely applicable experimental method, thermal denaturation has been used for a variety of purposes, including purifying proteins by selective denaturation of impurities and to study protein structure, folding, and stability.
• TDC Thermal denaturation curves
• DSC differential scanning calorimetry
• the DSC curves reflect the stability of many different structural domains, some sensitive to the binding of ligands and some not sensitive at all. Furthermore, denaturation may be initiated at many locations within the protein structure. Each of these processes has its own activation energy, which makes it the dominant process only within a narrow temperature range. As a consequence, depending on the scanning rate, the stability of a given domain may or may not be evident in the DSC curve. Furthermore, differential scanning calorimetry may see two or more protein denaturation steps where one would expect only a single transition.
• the present invention provides methods for identifying compounds that bind to target species (e.g., polypeptides including proteins, and polynucleotides including DNA and RNA). These methods involve the use of isothermal denaturation, preferably in combination with fluoresence detection methods. Significantly, the methods of the present invention involve automated methods suitable for HTS and UHTS. Ideally, the methods of the present invention are envisioned to be scalable to evaluate 10,000-60,000 compounds or more in a 24 hour period.
• target species e.g., polypeptides including proteins, and polynucleotides including DNA and RNA.
• the present invention couples fluorescence techniques with denaturation by isothermal methods to determine alteration of target (e.g., protein) stability by a bound ligand.
• target e.g., protein
• the denaturation and stabilization or destabilization of target species (e.g., protein targets) by ligands against isothermal denaturation is quantified by changes in fluorescence intensity.
• the present invention provides a high throughput screening method for identifying a test compound that binds to a target species.
• the method includes: incubating a plurality of test mixtures under isothermal denaturing conditions, each test mixture including at least one test compound (preferably, at least two test compounds, and more preferably, 2 to 10 test compounds) and at least one target species (preferably, only one target species is in any one test mixture), wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature (e.g., unfold) to a measurable extent.
• the method further involves detecting a denaturation signal of each target species in the presence of the at least one test compound; and comparing the denaturation signal of each target species in the presence of the at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions.
• the methods of the present invention can evaluate at least about 100 test mixtures per day. Preferably, such an evaluation occurs substantially simultaneously.
• the target species can be a polypeptide (e.g., protein) or a polynucleotide (e.g., DNA or RNA).
• the target species is a protein.
• the compound can bind to the target species either specifically (e.g., at a specific site or in a specific manner) or unspecifically.
• the binding can involve a variety of mechanisms, including covalent bonding, ionic bonding, hydrogen bonding, hydrophobic bonding (involving van der Waals forces), for example, or combinations thereof.
• Isothermal denaturing conditions refers to conditions effective to denature a target molecule at a fixed temperature. It can also involve defined conditions with respect to pH, ionic strength, cation concentration, etc., which are generally held constant for evaluation of various compounds for a given target.
• Denaturation signal refers to the signal produced by the target species upon being denatured.
• T m refers to the midpoint of the melting transition of the target as determined by differential scanning calorimetry.
• Reporter molecule refers to a separately added molecule such as a fluorescent dye or a covalently bonded reporter group attached to the target.
• FIG. 4 Time-dependency of the isothermal denaturation of TK in the presence of thymidine monophosphate (TMP) monitored by SYPRO Orange.
• TMP thymidine monophosphate
• the experimental denaturation conditions for monitoring the isothermal denaturation of TK by SYPRO Orange fluorescence are described in the Methods.
• the reaction mixture in 2 mL of buffer contained protein, 0.5 ⁇ M, SYPRO Orange, at a 20,000-fold dilution, and TMP at the indicated concentrations. TMP was present in the reaction mixture at the time of the addition of protein.
• the data were analyzed using Equation 11 and the solid lines represent the theoretical fits to the experimental data.
• FIG. 7 Time-dependency of the isothermal denaturation of TK monitored by CD.
• the data were analyzed using a first-order rate model and the solid lines represent the theoretical fits to the experimental data points.
• Figure 9 DSC Scan of Stromelysin.
• the thermal melting profile of stromelysin was determined as described in the Methods.
• Figure IOA Time-Dependency of the isothermal denaturation of
• Equation 11 Representative data in the presence of 1.5 and 5 ⁇ M PNU-143988 are also shown. The solid lines represent the theoretical fits to the experimental data points.
• the present invention is directed to the use of isothermal denaturation.
• the methodology can be used to screen for ligands to a wide variety of molecules, particularly proteins, including those with unknown function.
• the methods of the present invention eliminate the necessity of ramping temperatures up and down and should allow for much faster assay development and higher throughput in an HTS or UHTS automated environment.
• the technology should be easily expandable to looking for compounds that bind to RNA, DNA, ⁇ -acidic glycoprotein, and serum albumin, for example.
• Isothermal denaturation offers an attractive alternative method for monitoring denaturation (e.g., unfolding of a target species) and for the identification of binding ligands. It is amenable to HTS and UHTS. Furthermore, the denaturation process is easily controllable, reproducible, and independent of the heating rate.
• the choice of temperature used in isothermal denaturation can be determined by measuring the rate of denaturation of the target species at a series of temperatures (e.g., within a range of about 45°C to about 75°C). These measurements may be made, for example, using a fluorescent reporter molecule that binds to and reports conformational changes associated with the unfolding of the target molecule.
• denaturation signals can be monitored using UV absorbance, CD ellipticity, or by microcalorimetry studies with the target species, for example.
• a preliminary DSC scan is run to determine T m (midpoint temperature) of the target species in appropriate buffers that enhance the stability of the target over a long period of time as would be known to one skilled in the art.
• all components are maintained at one given temperature (preferably ⁇ about 0.2°C) which is chosen to produce a slow, easily monitored denaturation of the target protein. If the temperature of isothermal denaturation is too low, the kinetics are too slow. Generally, it is desirable to have a detectable amount of denaturing (e.g., unfolding) occur within about 60 minutes or less. If the temperature is too high, the kinetics are so fast that the test compound would not be able to stabilize the denatured target species resulting, for example, in too great an extent of unfolding. Too much unfolding can cause aggregation that could result in precipitation of the target. Furthermore, at too high a temperature, the test compound may not bind at all.
• one given temperature preferably ⁇ about 0.2°C
• the desired temperature for isothermal denaturing is equal to the T m value ⁇ about 10°C of the target species as determined by DSC. More preferably, this temperature is equal to or up to about 10°C less than the T m value of the target species.
• the target species preferably together with a suitable reporter molecule able to monitor its denaturation, is incubated in the presence and absence of the target species.
• the concentration of the compound and that of the reporter molecule are of comparable magnitude (preferably, no greater than about 1 ⁇ M), but may require the reporter molecule to be in excess relative to the target molecule, whereas the concentration of the test compound is in at least a 10-fold excess.
• the percent inhibition cutoff for a "hit" can be set prior to assay implementation, or determined statistically during or after all screening has been performed.
• Fluorescence techniques are rapidly becoming the detection methods of choice for HTS and UHTS.
• fluorescence molecules are used as the markers of choice. Coupling fluorescence techniques with denaturation by isothermal methods is attractive because in isothermal denaturation the quantum yield of an extrinsically added reporter molecule is dependent only on changes in protein folding and not on temperature effects. Further, any change in the fluorescence quantum yield measures binding of the reporter molecule to different denatured forms of the target species. Thus, alteration of target stability by a bound ligand should be easily detectable.
• the present invention demonstrates that isothermal denaturation can be used to determine if known competitive inhibitors/ ligands could bind to target species. The results are comparable to those obtained by other methods. The agreement of the denaturation kinetics from three different detection methods confirms that the same unfolding processes are being measured using the methods of the present invention.
• the fluorescence of the reporter molecule should preferably increase several-fold (preferably, at least about 2-fold) upon denaturation of the target. For proteins, this is typically accompanied by the exposure of the protein's hydrophobic regions.
• the reporter molecules should also preferably have low affinity for the native target; that is, the fluorescence of the native target/reporter molecule complex is linear over a wide concentration range or, preferably, does not bind to the native target at all so that it does not become a ligand itself.
• the reporter molecule should preferably have excitation and emission in the visible region where few compounds interfere, e.g., excitation at about 488 nm and emission at about 515 nm.
• Reporter molecules e.g., fluorescent dyes
• sources such as Molecular Probes (Eugene, OR) and fluoresce brightly when bound to hydrophobic regions of the target molecule.
• These include SYPRO Orange, SYPRO Red, Nano Orange, Nile Red, 1 -anilinonaphthalene-8- sulfonic acid (1,8-ANS), and dapoxylbutylsulfonamide (DBS) as well as other dapoxyl analogs.
• Nano Orange fluorescence provides an ultra-sensitive dye for quantification of proteins in solution with a linear fluorescence range of about 10 nanograms/milliliter (ng/mL) to about 10 micrograms/milliliter ( ⁇ g/mL) with a very low background fluorescence.
• SYPRO Orange and SYPRO Red are used for gel staining with sensitivity as good as silver staining.
• the basis for the increase in fluorescence of the dyes with protein denaturation is their binding to newly exposed hydrop lobic sites.
• 1,8-ANS has been used extensively for many years to monitor the unfolding of proteins; however, its quantum yield when bound to the denatured protein is much lower than those of the dyes discussed above and, thus, would require the use of large quantities of protein and reporter molecule in the assays.
• DBS is a relatively new, solvatochromic dye whose fluorescence emission may shift as much as 100 nm upon changing the environment. Due to its lower excitation and emission wavelengths, however, it is less desirable than Nano Orange, SYPRO Orange, or SYPRO Red for HTS. Any fluorescent reporter molecule whose emission intensity increases or decreases when bound to a desired target species can be used for isothermal denaturation.
• the affinity of a fluorescent reporter molecule toward a target species can be determined by measuring the fluorescence of a given concentration of the reporter molecule in the presence of increasing concentrations of the denatured target species and the native target species. Knowing the affinity then allows one to optimize the concentration of the fluorescent reporter molecule relative to the target species.
• target species labeled covalently with a pair of fluorophores one of which quenches the fluorescence of the other. Because unfolding of the target species changes the intermolecular distances between the two fluorophores, the denaturation is accompanied by changes in fluorescence. By labeling the same target species at specific sites, the denaturation at different structural regions can be monitored.
• fluorescence techniques particularly dye binding resulting in fluorescence enhancement are the detection methods of choice, other techniques can be used in the methods of the present invention.
• the experiments can be performed in the presence of a chaotrope, such as urea, guanidine hydrochloride, organic solvents, or any other reagents that promote protein denaturation without unduly interfering with binding of the reporter molecule with the target species.
• a chaotrope such as urea, guanidine hydrochloride, organic solvents, or any other reagents that promote protein denaturation without unduly interfering with binding of the reporter molecule with the target species.
• the methods of the present invention can be carried out in a multi -reservoir sample holder, such as a microtiter plate.
• a multi -reservoir sample holder such as a microtiter plate.
• all components but the target species are added and the multi-reservoir sample holder is held at the appropriate temperature for a period of time.
• the sample holder is preferably transferred to a station where the target species is added to all reservoirs, preferably simultaneously.
• the multi-reservoir sample holder is typically sealed prior to addition of any components.
• a microtiter plate can include a covering that is made of a plastic sheeting which seals the plate but is scored in such a way that a microtiter tip easily penetrates it but that it re-closes after tip removal.
• the sample holder is either transferred immediately to an appropriate detector for reading the denaturation signal or to an incubator for holding until detection is desired. All steps can be performed either manually or by robot as desired.
• a commercially available Zymark/ Zymate PCS system (Zymark Corp., Zymark Center, Hopkinton, MA) equipped with a Rapid Plate module, jacketed carousel, 10-plate incubator system interfaced with a fluorescent plate reader can be used.
• This system can process 96- and 384-well microtiter plates and can be adapted for use in the isothermal denaturation method of the present invention.
• the 10- plate incubator can be modified with heating elements such as Watlow flexible flat mat heaters for sample incubation.
• the temperature of the incubator can be further controlled by the use of a circulating waterbath.
• the Zymark/Zymate system includes a jacketed carousel that can be modified to include a temperature controlled humidifier and fan internally, and heat lamps externally, to assist in temperature control and to reduce loss of sample volume in the microtiter plates.
• the Zymark/Zymate system also includes a pipetting station (Rapid Plate Module) that can be modified to include a heating block and heat lamps, for example.
• a BMG POLARstar microplate reader BMG Labtechnologies, Inc., Durham, NC
• a circulating waterbath e.g., from about -20°C to about 90°C. This system is automated using robotics and computer software, which can be modified to allow for the samples to experience isothermal conditions.
• TK thymidylate kinase
• stromelysin thymidylate kinase
• the time dependencies were all consistent with a reaction scheme of two consecutive first-order reactions. That is, the kinetics of denaturation for both proteins were best described by a biphasic model. Thus, only two of the probably many steps are rate limiting. It is apparent that a significant amont of information of the kinetics of the unfolding processes are provided by the fluorescence measurements.
• the dependence of the rate constants on ligand concentration was analyzable in terms of a binding isotherm, reflecting the stabilizing effect of the protein ligand complex.
• the method was validated by comparing its results with those obtained by steady-state fluorescence spectroscopy, circular dichroism, and UV spectrophotometry.
• the corresponding rate constants calculated from the results of the several analytical detection methods were comparable.
• the rate constants of both steps were dependent upon the binding of active-site ligands.
• the dissociation constants represent affinities of the ligands at the melting transition temperature.
• the affinity constants i.e., "dissociation constants" at physiological temperatures can be determined by extrapolation from measurements at two different temperatures.
• SYPRO Orange, SYPRO Red, Nano Orange, l-anilinonaphthalene-8- sulfonic acid (1,8-ANS), and dapoxylbutylsulfonamide (DBS) were purchased from Molecular Probes Inc., Eugene, Oregon. Thymidine monophosphate
• PNU-143988 has the following structure: B. Proteins
• S. aureus thymidylate kinase was cloned by Human Genome Sciences and purified by affinity chromatography using Ni 2+ -NTA columns purchased from Qiagen (QIAGEN Inc., Valencia, CA).
• TK the isothermal denaturation took place in a 5 millimolar (mM) Tris buffer, pH 7.80, containing 0.5 mM ⁇ -mercaptoethanol and was measured at a temperature of 53°C, with the exception of the validation experiments of the robotic assay which were carried out at 52°C.
• aureus uridylate kinase (UK) was cloned by Human Genome Sciences and purified by affinity chromatography using Ni -NTA columns (QIAGEN Inc., Valencia, CA). UK has a transition midpoint, T m , of 45.5°C as determined by DSC using a buffer of 50 mM TRIS, 500 mM NaCl, 10% glycerol, and 5 mM ⁇ -mercaptoethanol, pH 7.8. Validation experiments for isothermal denaturation were performed at the T m , 41 °C, in pH 7.5 buffer composed of 50 mM Tris, 200 mM KC1, 10% glycerol, and 5 mM ⁇ - mercaptoethanol.
• Stromelysin was cloned and purified as described by Finzel et al., Prot. Sci.. 7, 2118-2126 (1998). It has a T m of 75°C as determined by DSC. This temperature was chosen for the following isothermal denaturation experiments.
• the buffer system for stromelysin consisted of 10 mM imidazole, 2.5 mM CaCl 2 , 5 micromolar ( ⁇ M) ZnCl 2 , pH 6.50.
• DMSO dimethylsulfoxide
• Stock solutions for all ligands were prepared in dimethylsulfoxide (DMSO) unless noted otherwise. Whenever the water solubility was high enough, secondary stock solutions were made in the buffer system for the particular protein; otherwise, diluted stocks were prepared in DMSO. A small aliquot of ligand solution, typically 10 ⁇ l or less, was added to buffer and equilibrated at the appropriate temperature in the cell before addition of the protein. Control experiments assessed the effect of added DMSO on protein denaturation. In some cases 0.1% CHAPS (weight/volume percent, (3-[(3- choloamidopropyl)-dimethylammonio]-l-propanesulfonate) was added to the reaction mixture to counteract the effects of DMSO.
• CHAPS weight/volume percent, (3-[(3- choloamidopropyl)-dimethylammonio]-l-propanesulfonate
• a photon counting ISS K2 spectrofluorometer in the ratio mode was used (ISS Inc., Urbana, Illinois). The temperature was maintained within 0.2°C throughout the experiments by means of a Polysciences programmable temperature controller (Polysciences, Niles, IL). Emission was observed on the filter channel using the following emission filters: 530 nanometer (nm) bandpass filter for Nano Orange, 590 nm cut-off for SYPRO Orange, 630 nm cut-off for SYPRO Red and Nile Red, and 470 nm cut-off for 1,8-ANS and dapoxylbutyl-sulfonamide.
• fluorescence measurements were acquired by a BMG POLARstar microplate reader (BMG Labtechnologies, Inc., Durham, North Carolina). Temperature was controlled with a circulating waterbath. This instrument best detected Nano Orange using a 485 nm center wavelength, 15 nm bandpass excitation filter and a 580 nm center wavelength, 12 nm bandpass emission filter.
• the time-dependencies of the fluorescence of extrinsic dyes during isothermal denaturation were monitored as follows.
• the test dye was added to a stirred cuvette containing 2 milliliters (mL) of buffer that had been thermally preequilibrated at the desired temperature.
• a dye baseline was recorded for 45 seconds and the denaturation reaction initiated by the addition of a small aliquot of the protein stock solution. In this way, the protein reached the temperature of denaturation virtually instantaneously.
• the protein was kept at the denaturation temperature in the absence of dye and the reporter molecule added at the end of the reaction. In the latter experiments, the fluorescence increase associated with the addition of dye was always instantaneous. In order to be certain that under the conditions of the experiment the dye itself would not complex a significant fraction of the native protein, a fixed amount of dye was titrated with increasing amounts of protein and showed that the fluorescence increase did not reach saturation.
• the three tryptophan residues of stromelysin are buried in the active site region and as such are sensitive reporters of the unfolding of the protein.
• the isothermal denaturation of this protein was also measured by changes in the intrinsic tryptophan fluorescence.
• the protein was added to buffer equilibrated in a cuvette at the temperature of isothermal denaturation.
• the excitation wavelength was 293 nm and the emission was monitored using a 320 ⁇ 10 nm bandpass filter.
• DSC for stromelysin and TK was performed using an MC-2 differential scanning calorimeter from Microcal, Inc. (Northampton, MA).
• stromelysin the 1.2 mL sample cell of the calorimeter was filled with 150 ⁇ M enzyme in a pH 6.50 buffer containing 10 mM imidazole, 2.5 mM CaCl 2 , and 5 mM ZnCl 2 .
• TK the calorimeter cell was filled with 15 ⁇ M enzyme in a 50 mM Tris- HC1 buffer, pH 7.70, containing 0.50 M NaCl, 10% glycerol, and 5 mM 2- mercaptoethanol.
• the reference cell was filled with the same buffer.
• the solutions were degassed for 5 minutes prior to scanning from 25°C to 80°C at a rate of 1 °C/minute.
• Baseline scans collected with dialysate buffer in the sample cell, were subtracted from the protein scans and the resulting data converted to unit protein concentration.
• the Y-axis of the instrument was calibrated using standard electrical heat pulses and the temperature scale was calibrated using n- octadecane and w-hexatriacontane which melt at 28.2°C and 75.9°C, respectively.
• Circular Dichroism (CD) spectra were measured using a Jasco J-715 spectropolarimeter (Jasco Corp., Easton, MD) and a cylindrical quartz cell with a pathlength of 0.1 centimeter (cm) thermostated to within 0.1 °C by a Haake D8 circulating water bath (Haake Gmbh, Düsseldorf, Germany).
• the concentration of the protein was chosen on the basis of its molar ellipticity and fell usually in the range of about 1 ⁇ M to about 20 ⁇ M. Solutions of protein or protein with 10- to 100-fold excess of ligand were prepared prior to injection into the cell.
• Each solution was first scanned at 22°C from 178 nm to 260 nm with a response of 0.25 second, scan speed of 100 nm/minute, resolution and bandwidth of 1.0 nm and 5 accumulations.
• the cell was then rapidly heated to the temperature of isothermal denaturation and the time dependency of the ellipticity at 222 nm was monitored.
• Dichroism was sampled with a bandwidth of 1.0 nm at 0.5 millisecond (msec) intervals and accumulated for 16 seconds. Data were stored every one second as the running average of the 16 second bundles. After the time scan, a wavelength scan was performed at the same temperature.
• Cells and buffer solutions with and without ligand were routinely checked for absorbance and dichroism. Cells were thoroughly cleaned as described above and rinsed with distilled water and ethanol between experiments.
• UV absorbance was measured using a Perkin-Elmer Lambda 40 UV-Vis dual-beam spectrophotometer (Perkin-Elmer Corp. Norwalk, CT).
• a capped 1.0 cm pathlength quartz cuvette filled with buffer was placed in the reference beam.
• a capped 1.0 cm path length quartz cuvette containing 1.5 mL of degassed buffer or buffer plus ligand — less the volume of protein solution to be added — was placed in a thermostated cell holder and equilibrated at the temperature of isothermal denaturation. The temperature was maintained within 0.1 °C using a Neslab Exacal EX200 circulating water bath (Neslab Instruments, Inc. Portsmouth, NH).
• a 1-5 ⁇ L aliquot of the protein stock solution was added to the cuvette containing buffer or buffer plus ligand to yield a final volume of 1.5 mL and protein concentration near 0.5 ⁇ M.
• the cuvette was recapped and absorbance at 280 nm was measured every 1 second for up to 30 minutes with a bandwidth of 2 nm and response of 0.5 second.
• the effects of several ligand concentrations were examined ranging from about 0.5 ⁇ M to ⁇ bout 400 ⁇ M.
• Cuvettes were thoroughly cleaned as described above with nitric acid and with 10% methanol and rinsed with distilled water and ethanol between experiments.
• Y Y 0 + A Y . (l - . " t "' ' ) Equation 1
• Y is the experimentally measured signal
• Y 0 is the background signal
• ⁇ Y is the total change in signal associated with the denaturation process
• t is time
• k ex p is the experimentally measured apparent first-order rate constant.
• the kinetics of denaturation displayed biphasic kinetics, often with a distinct induction period. Those time courses were analyzed according to the reaction scheme of two consecutive first-order reactions:
• Equation 4 Equation 4
• Robotic Assay A library of compounds was tested in a high throughput screening mode in 96-well microtiter plate format with single compounds per well at the temperatures described above. The optimal dye and optimal ratio of dye to protein for TK and UK was assessed. Each microtiter plate contained 88 individual compounds and eight control wells that intially contained only buffer plus dye (no compound). These assay plates were manually sealed with a plastic 96-well microplate seal (Tomtec, Inc., Hamden, Connecticut). These seals were scored for easy entrance of pipet tips, followed by reclosure after pipet tip exit. The remaining steps were carried out robotically as follows.
• Assay plates were deposited into the temperature/humidity-controlled incubator for an initial incubation period, typically 60-90 minutes, which equilibrated the assay plate to the desired assay temperature.
• a fluorescence measurement,Tesse was then taken by the BMG POLARstar to establish the lower bound of the assay. This fluorescent reading for those wells containing compounds plus buffer plus dye was used to ascertain the effect of the compounds themselves.
• assay plates were moved to the RapidPlate liquid dispensing unit. Protein was added from a plate reservoir to assay plates located on a modified heated plate position.
• assay plates were transported back to the temperature/humidity-controlled incubator for a defined time at the assay temperature, typically 30 minutes.
• a second fluorescence measurement, Tf was taken by the BMG POLARstar.
• the control wells (assay buffer plus dye plus protein) defined the upper bound of the assay.
• a comparison of the fluorescent measurement for the wells containing compound (plus assay buffer plus dye plus protein) compared to the control well reads at T, and T f defined which compounds bound to and stabilized the protein of interest.
• the temperature at which a given protein undergoes denaturation at an easily measurable rate was determined prior to the ligand binding studies. The rates were most appropriate for measuring methods at temperatures slightly below or at the thermal transition temperature (T m ) as measured by differential scanning calorimetry. To determine which fluorescent dye produces the largest signal for a given protein, a preliminary isothermal denaturation experiment at T m was performed with each dye at a concentration of 1 ⁇ M and the protein at 0.5 ⁇ M. The spectral properties of all dyes tested are given in Table 1. Due to their long excitation and emission wavelengths, Nano Orange, SYPRO Orange, and SYPRO Red are attractive for use in HTS format.
• TK thymidylate kinase
• Figure 1 The thermal scan for thymidylate kinase (TK), shown in Figure 1 , reveals a T m located at 53°C, the temperature which was then chosen for all subsequent isothermal experiments. Upon cooling and reheating of the protein, no observable peak was found (results not shown), which indicated that this protein had denatured irreversibly.
• Fluorescence, i f igure 2 shows the results of preliminary experiments where the time-depend _nt fluorescence changes for each dye were measured in the presence of thymidylate kinase denaturing isothermally at 53°C.
• FIG. 4 shows representative time courses of SYPRO Orange fluorescence occurring during the isothermal denaturation of TK in the presence of increasing amounts of TMP, which is a specific ligand for the enzyme.
• TMP which is a specific ligand for the enzyme.
• Equation 11 the model of two consecutive first-order reactions with identical rate constants, Equation 11 , was the most consistent with the data.
• the agreement of the experimental points with the theoretical curves calculated with the best-fit parameters and Equation 11 is shown in Figure 4.
• the slope of the early, linear portion of the time course is also proportional to the first rate constant and, thus, either can be used to analyze the influence of TMP concentration on the denaturation rates.
• Figure 5 shows the dependency of these two parameters on ligand concentration. The curves were analyzed using a nonlinear least squares method and equations corresponding to a variety of models relating denaturation rates to occupancy of the ligand binding site.
• Circular dichroism scans of the native protein and of its denatured form show that a significant change in the CD spectrum accompanies the unfolding (Figure 6). For example, there was a 45% loss in intensity of the ⁇ -helical signal at 222 nm. Consequently, the time-dependency of the changes in molar ellipticity was monitored continuously at 222 nm. As illustrated in Figure 7, the ellipticity at 222 nm increased rapidly and reached a maximum after about 10 minutes. Addition of 25 mM TMP, a natural ligand, greatly decreased both the rate and magnitude of the increase in ellipticity. The data were fully consistent with a simple first-order reaction, thereby indicating that the major changes in ellipticity are produced by the first of the two consecutive unfolding steps.
• the rate constants obtained by the three techniques are compared in Table 2. Due to spectral interference, the effects of the presence of TMP on the changes in hyperchromicity were not tested. It appears that the physical properties measured by all these methods do undergo a significant change during the first rate-limiting step. There was good agreement between the rate constants of the first step, k ⁇ , indicating that all three methods are measuring the same process. On the other hand, the second step observed by SYPRO Orange does not produce any changes in hyperchromicity and the second, very slow step observed by hyperchromicity does not have any changes in SYPRO Orange fluorescence associated with it.
• the intrinsic tryptophan fluorescence of stromelysin is particularly sensitive to ligand-induced conformational changes, and, presumably, to denaturation of the active-site region.
• Figure 10B there was a rapid, biphasic loss of the tryptophan fluorescence intensity over the time course of the isothermal denaturation at 75°C.
• the time-dependency of the decay of the tryptophan fluorescence was most consistent with a model of two consecutive first-order reactions with identical rate constants (Equation 11). This model yielded rate constants that were in excellent agreement with those measured by the fluorescence increase of SYPRO Orange under the same conditions (Table 3).
• Two parameters derived from the fluorescence kinetic curves measure the rate of denaturation, namely, the slope at the inflection point, and the first- order rate constant.
• the values of both parameters decreased in the presence of inhibitors in a saturable, dose-dependent manner, as shown in Figure 11 for the case of fluorescence measurements.
• the concentration dependencies were consistent with a model where binding of the inhibitor results in a drastic decrease in the rate of denaturation.
• Validation of the HTS system was furthered by the isothermal denaturation of TK and UK in the presence of a subset of compounds with an increased potential of containing protein ligands since these compounds had inhibitory activity in activity assays. This validation test was performed as described with 10 ⁇ M final compound concentration. Using the Tj and T f measurements, percent inhibition was calculated. True actives were determined by using three standard deviations from the mean of the assay plate controls as the active cutoff value. Results are shown in Table 4. Control experiments were also conducted. Compounds with intrinsic spectral properties including fluorescence and quench were observed in these experiments.
• S. aureus FemA (Ehlert et al., J. Bacteriol.. 179, 7573-7576 (1997) and Tschierske et al., FEMS Microbiol. Lett.. 153. 261-264 (1997)) is a protein presumably involved in cell wall biosynthesis and thus provides an attractive target as a potential antibacterial.
• the protein is expressed with a 6-his tag so that it can be purified with Qiagen Ni 2+ -NTA columns as for thymidylate kinase described above.
• a pH sufficiently removed from the isoelectric point (pi value) is chosen for the buffer in which the protein is solubilized; in addition appropriate ionic strength and cations are used so that maximal structure can be obtained as monitored, e.g., in CD ellipticity studies.
• this protein Since this protein has no known biochemical function, isothermal denaturation provides an ideal way to discover compounds that bind to this protein. It is believed that this protein exhibits multi-phasic kinetics similar to those observed with thymidylate kinase and stromelysin.
• the T m value is determined by differential scanning calorimetry studies. The detailed kinetic pathway of denaturation must first be determined by fluorescence, absorbance, and/or another physical method as described above. The optimal dye and optimal ratio of dye to protein is rapidly assessed in a 96-well microtiter plate format. The fluorophore used is SYPRO Red.
• a library of compounds (> 100,000) are tested in a high throughput screening mode in 96-well microtiter plate format with single compounds per well at or, at most, 5°C below the T m value.
• Each microtiter plate contains 88 individual compounds.
• eight control wells exist in the plate that intially contain only buffer plus dye (no compound).
• Tj read the microtiter plate containing compounds plus control wells are read (Tj read) which establishes the lower bound of the assay. This fluorescent reading for those wells containing compounds plus buffer plus dye is used to ascertain the effect of compounds themselves.
• T f read After a time at the assay temperature, e.g., 30 or 60 minutes, another fluorescent measurement is performed (T f read).
• the control wells (assay buffer plus dye plus protein) define the upper bound of the assay.
• a comparison of the fluorescent values for those wells with compound (plus assay buffer plus dye plus protein) compared to the fluorescent values of the control wells at Tj and T f defines which compounds bind to and stabilize the protein of interest, which in this example is S. aureus FemA. Only those compounds that demonstrate stabilization in a subsequent repeat experiment are pursued as potential ligand binders.
• a compound could give enhanced fluorescence because it: 1) is a hydrophobic compound that could bind dye; 2) may have intrinsic fluorescence at the wavelengths used; 3) could form micelles; or 4) could be a denaturant, destabilizer, etc.
• a compound could have binding activity but exhibit lower than expected results because the compound adsorbs light (quenches) at the wavelengths tested.
• compound plus dye plus buffer at the assay temperature are read before the addition of protein. Any compounds that have intrinsic fluorescence, quench, fluorescence enhancement or fluorophore sequestration can thus be identified.
• an additional study is performed after the screen has been performed. The effect of compound on dye binding to the target molecule is performed at ambient temperature. Any compound which is a denaturant demonstrates enhanced binding of the fluorescent dye to the protein even when no thermal denaturation of the protein occurs. Compounds of this type would only be of interest if they exhibited this property for a specific protein (target) and did not affect two or more proteins (targets) in this manner.
• compound mixtures can be tested.
• a subset of the entire library that contains mixtures of eight compounds per microtiter plate well are used.
• the assay is carried out as described above.
• the compounds for those mixtures that demonstrate the appropriate results are then identified and tested individually. In this case positive results could occur in mixtures but not for individual compounds because the results could be additive, or more likely, synergistic. Consequently, if assays with individual compounds are not active, permutations of mixtures can be tested to determine which combination gives the original screening result.
• subsetsof a libraryor "sublibraries,” based upon rationale criteria can be tested.
• the advantage of using a sublibrary(ies) is to accelerate the discovery of a useful compound from high throughput screening.
• S. aureus Unknown Gene Product is utilized, S. aureus Unknown Gene Product.
• This protein is an essential gene product for this organism.
• its biological/biochemical function is unkown; and although searching of genomic database identifies similar genes in other micro-organisms, they also have no known biological or biochemical function.
• constructs can be engineered placing a 6-his tag at either the amino- or caboxyl-terminal end of the protein and purified as described for the other 6-his tagged proteins described. Maximal structure under given experimental conditions as monitored, for example, by CD can be obtained, similar to the femA protein described above.
• dissimilarity sublibrary is generated by a dissimilarity search in which compounds are sorted on their structural/chemical properties. The most dissimilar compounds are selected but, similutaneously, they represent the diversity of the entire library. Compounds identified that stabilize this protein target in isothermal denaturation studies are tested further in their own right. In addition, compounds in the library with similarity to these ligand-binders can be selected from the entire library by using computer search programs. These are also tested. In this way the active compounds could potentially be identified by screening only a limited subset of the entire compound collection.
• RNA of interest can be synthesized or purchased commercially and assembled into duplex DNA in the proper order. The assembled DNA can then be inserted into an expression system (e. g. MEGAScript from Ambion) to generate an RNA of interest. Alternatively, if the RNA of interest is sufficiently small, the oligos can be constructed to contain an appropriate promoter such that in vitro transcription can be done without any cloning and expression steps. Isolation of RNA can be obtained by protocols known to anyone skilled in molecular biologic arts.
• a T m can be determined experimentally with DSC.
• extrinsic fluorescent dyes that can be used to monitor the transition from an ordered to a disordered RNA structure include SYBR Green, SYBR Greenll, Pico-Green, and TOPRO, YOYO, etc.
• RNA molecules that can be used to demonstrate this approach include: 1) HIV-1 tar 47-86 (Mei et al., Biochemistry. 37,14204-14212 (1998)); 2) RNA aptamer J6fl (Cho et al., Biochemistry, 37, 4985-4992 (1998)); and 3) A-site of 16s rRNA (Wong et al.. Chemistry and Biology. 5. 397-406 (1998)).
• Ligands known to bind to these respective RNA molecules are: 1) Neomycin, other aminoglycoside antibiotics, and other compounds (Mei et al., Biochemistry, 37, 14204-14212 (1998)); 2) tobra ycin ((Cho et al., Biochemistry. 37, 4985-4992 (1998)); and 3) Kanamycin and other aminoglycides (Wong et al., Chemistry and Biology. 5, 397-406 (1998)).
• ligands for proteinaceous targets stabilize their structures under isothermal conditions
• these known ligands stabilize their cognate RNA molecules under similar conditions.
• a large collection of compounds can be tested in high throughput screening to determine whether any might bind to, and stabilize, these nucleic acid structures under isothermal denaturation conditions. These compounds can be tested singly or as combinations of several compounds.
• one skilled in the art could also monitor these changes using UV hyperchromicity or capillary electrophoresis.

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